PROTEIN PROFILING IN CENTRAL NERVOUS SYSTEM FROM GALECTIN-3 KNOCKOUT MICE INFECTED WITH Toxoplasma gondii.
1,2Rosa, J.C.; 2Bernardes, E.S.; 1,2Gimenez, M.; 1,2Souza, V.C.O.; 2Ruas, L.P.; 2Fermino, M.L.; 2Roque-Barreira, M.C.
1Centro de Química de Proteínas e 2Depto. de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. Ribeirão Preto/SP 14049-900.
Recent studies in galectin-3 have revealed that it is expressed in a variety of cell types in the immune system, constitutively or in response to microbial invasion. These studies implicate galectin-3 in both innate and adaptive immune responses, where it participates in the activation or differentiation of immune cells. We have previously described that galectin-3 knockout mice (gal3–/–) infected with Toxoplasma gondii mount a higher Th1-polarized response, which was characterized by a higher ratio of T. gondii-specific IgG2a/IgG1 and increased levels of IL-12. In addition, we identified CD11c+ dendritic cells as responsible for the increase in IL-12 production, and consequently for the higher Th1 response in these mice (Bernardes et al., American Journal of Pathology, in press). In the present work, we investigated protein profiling expression in central nervous system (CNS) of gal3–/– mouse per-orally infected with twenty cysts of the ME49 strain of T. gondii compared to uninfected gal3–/– mouse CNS. After 14 days of infection, the mice were euthanized and samples of the CNS were collected. Proteins were prepared by tissue homogenization in lysis buffer (Tris-HCl, triton-X-100, protease inhibitor), TCA/acetone clarification, and ressuspended in 2D electrophoresis buffer. Proteins were separated by isoelectric focusing in IPG 3-10NL (7cm) as first dimension and 12% homogeneous gel of poliacrylamide SDS-PAGE as second dimension. Gels were stained by colloidal coomassie blue (CCB) and scanned images were analyzed using the ImageMaster 2D Platinum v.5.0 software. Image analysis detected 239 spots on gel of infected-mouse and 372 spots in uninfected mouse and after gel spot matching 18 spots were selected as having differential expression between uninfected and infected mouse. Five spots were detected in lower and 13 spots in higher level of expression in infected gal3–/– mice. Spots were digested with trypsin and analyzed by MALDI-TOF-MS. Most of proteins were identified as part of metabolism like enolase and aldolase, re-arrangements of cytoesqueleton as non muscular cofilin, and 5 of them were unknown proteins. These preliminary results indicate that protein profiling based on proteomic approach may collaborate to shed some light on the role of galectin-3 during infection by T. gondii. Supported by FAPESP, CNPq, FAEPA.
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